This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. ABSTRACT: Electron microscopic study of frozen-hydrated biological material avoids the necessity for chemical fixation, dehydration, and staining, and thus provides a view of the specimen in a """"""""near-native"""""""" state. The water in biological specimens must be frozen in """"""""vitreous"""""""" or amorphous form in order to avoid nanometer-scale damage to the specimen due to ice crystal formation. For tissue, the preferred method is high-pressure freezing, due to the depth of good freezing that can be obtained. The frozen tissue must be maintained below the de-vitrification temperature (~-140?C) throughout ultramicrotomy and microscopy. Steady progress has been made over the past two decades in cutting frozen-hydrated sections. Although it remains a challenging task, improved cryo-ultramicrotomes and diamond knives, together with the collective experience of the few laboratories engaged in this work, provided a good starting point for investigators wishing to make use of frozen-hydrated sections. In April, 2002, we were the first laboratory to obtain electron tomograms of frozen-hydrated sections (from high-pressure frozen rat liver tissue). The most important finding was that the interior of the section was free of surface artifacts, thus good 3-D information could be obtained. + Hsieh, C.-E., Marko, M., Frank, J., and Mannella, C.A. 2002. Electron tomographic analysis of frozen-hydrated tissue sections. J. Struct. Biol. 138:63-73. We did subsequent work in three areas: improvements in high-pressure freezing, comparison of frozen-hydrated and freeze-substituted material, and improvements in section attachment to grids. Rat liver tissue was frozen using needle biopsy kits, with which it was possible to freeze tissue within 40 sec of blood flow cessation. Some tissue was freeze-substituted and embedded in plastic. The main differences between the two techniques related to the relative contrast of cellular components. Tomograms of frozen-hydrated sections showed excellent structural preservation, which correlated with good sectioning quality, yielding sections with few surface artifacts (crevasses). We investigated means to reduce the irreversible compression that occurs in the sectioning direction. Based on recommendations in the literature, we started tested both an oscillating 35? cryo diamond knife and a 25? diamond knife, but no improvement in compression was seen in initial tests. One of the major problems with tomography of frozen-hydrated sections is poor attachment of the sections to the gird. This is due in large part to lack of section flatness, which we documented by low-magnification stereo pairs. We found that sections could be attached to Quantifoil grids by use of a glass press tool, and the thinner Quantifoil grids are advantageous for tomography because they allow more open area at high tilt. We also found that the use of molybdenum grids reduced wrinkling of the carbon film, and may aid in section attachment. We identified strategies for identifying suitable grid areas for tomography, thus increasing the yield of successful tomograms. We summarized the findings of the last reporting period in two publications: + Hsieh, C.-E., Marko, M., Leith, A., Mannella, C.A. and Frank, J. (2006) Towards high-resolution three-dimensional imaging of native mammalian tissue: Electron tomography of frozen-hydrated rat liver sections. J. Struct. Biol. 153(1):1-13. + Marko, M., Hsieh, C.-E., and Mannella, C.A. (2006) Frozen-hydrated sections for electron tomography of cells and tissue. In: Ed. J. Frank, Electron tomography of cells and tissue, Springer. We attended the third international meeting on sectioning of vitreously-frozen specimens, held at Schloss Hohenkammer, near Munich, Germany on May 21-23, 2006. There, we learned of progress in other labs regarding the section attachment problem. One approach consisted of application of markers (in the form of quantum dots) directly on sections. Another approach used an electrostatic charge to help attachment of sections to a support film. We are investigating both approaches, realizing the limitations of each, especially in the case of thick sections and sections with crevasses that may deform during tilt-series collection. Because of this work, we are taking a hiatus from the collaborative work with Dr. Toh-Ming Lu of RPI. We had made preliminary investigations of functionalized coatings for TEM grids to aid in section attachment, but results were inconclusive and we decided that the RPI effort should be concentrated on TRD2. We presented the work on tomography of frozen-hydrated sections at several venues, mostly combined with our work on FIB-milling (see below). One talk concentrated mainly on cryo-ultramicrotome sections: + Hsieh, C.-E., Mannella, C., Ting, C., Stokes, D., Frank, J., Marko, M. (2006) Cryo electron tomography of frozen hydrated sections of tissue and cells. Proc. 16th International Congress on Microscopy H. Ichinose and T. Sasaki, eds. 2:691. Related to cryo-tomography, although not frozen-hydrated sections, the following book chapter was published: + Marko, M. and Hsieh, C.-E. (2006) 3-D cryo-electron microscopy of cells and organelles. in: Ed. J. Kuo, Meth. Mol. Biol. 117.
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